High expression of the anti-apoptotic Bcl-2 family protein Mcl-1 is a critical survival mechanism in human multiple myeloma (MM) cells, and a key barrier to apoptosis induced by the Bcl-2/-xL antagonist ABT-737. De novo expression of the short-lived protein Mcl-1 is controlled transcriptionally by CDK9, a catalytic subunit of the PTEF-b transcription elongation complex (CDK9/cyclin T) that phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) to promote elongation of nascent transcripts. Thus, approaches targeting CDK9 may enhance ABT-737 efficacy in MM cells.
To test this hypothesis, we employed the pan-CDK inhibitor roscovitine and a highly-selective CDK9 inhibitor (CDK9i) in drug-naïve and drug-resistant MM cell lines, as well primary plasma cells.
Co-administration of non- or sub-toxic concentrations of roscovitine or CDK9i interacted in a highly synergistic manner with ABT-737 (e.g., 150-300 nM) to induce apoptosis in U266, RPMI 8226, H929, OPM-2, and MM.1S MM cells, as well as IL-6-depedent ANBL-6 and KAS 6/1 cells. In each case, Combination Index values were significantly less than 1.0. These regimens were fully active in various drug-resistant MM cells, including U266/VR and OPM-2/VR bortezomib-resistant cells, RPMI 8226/RR lenalidomide-resistant cells, MM.1R dexamethasone-resistant cells, RPMI 8226/LR5 melphalan-resistant cells, and RPMI 8226/Dox40 doxorubicin-resistant cells. They also effectively killed MM cells in the presence of stromal cells or growth factors (i.e., IL-6, IGF-1, BAFF, and APRIL). Co-administration of roscovitine significantly enhanced ABT-737 lethality in primary CD138+ MM cells, while largely sparing CD138− bone marrow cells. Notably, the majority of MM cell lines exhibited high expression of cyclin T1 rather than cyclin T2a/b, in association with constitutively-activated CDK9, manifested by high levels of phosphorylation of CDK9 and RNA pol II CTD at Ser2 and 5. Exposure of MM cells to either roscovitine or CDK9i strikingly reduced RNA pol II CTD phosphorylation at both sites (particularly Ser5), but did not affect CDK9 and cyclin T1 levels. Whereas treatment with ABT-737 alone led to a modest but clear increase in Mcl-1 expression, roscovitine or CDK9i completely blocked this event and substantially down-regulated basal Mcl-1 expression. Notably, similar phenomena occurred in U266/VR cells, which displayed higher basal levels of Mcl-1, an important candidate resistance mechanism, compared to parental cells. Moreover, knock down of Mcl-1 by stable transfection with shRNA dramatically increased U266 sensitivity to ABT-737. Co-treatment of MM cells with ABT-737 and roscovitine or CDK9i did not affect expression of other anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL, but induced a Bcl-2 cleavage fragment. Expression profiling of BH3-only proteins in MM cells co-exposed to roscovitine and ABT-737 revealed a modest or a marked increase in expression of Bim (both EL and L isoforms) and Noxa (particularly a ∼27 kDa species), respectively, accompanied by a clear decrease in Puma levels. No changes in expression of other BH-3-only proteins (e.g., Bid, Bad, Bik, BMF, Hrk) were observed. Interestingly, knock down of Bim by stable transfection with shRNA dramatically diminished roscovitine/ABT-737 lethality, whereas Noxa knock-down by shRNA only partially protected cells from this regimen. In contrast, both shRNA approaches substantially protected U266 cells from bortezomib lethality.
Collectively, these findings provide a mechanistic framework for targeting transcription by CDK9 inhibitors to diminish ABT-737-mediated Mcl-1 upregulation and to downregulate basal Mcl-1 levels, resulting in highly synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents. They also suggest that the BH3-only protein Bim may be required for apoptosis triggered by this strategy, while up-regulation of Noxa may amplify MM cell killing in this setting.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.